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- EMDB-22366: In situ structure of GABAA receptor pair -

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Basic information

Entry
Database: EMDB / ID: EMD-22366
TitleIn situ structure of GABAA receptor pair
Map datareconstruction GABA receptor pair
Sample
  • Complex: gamma-aminobutyric acid receptor
Biological speciesRattus norvegicus (Norway rat)
Methodsubtomogram averaging / cryo EM
AuthorsLiu YT / Tao CL / Zhang X / Xia W / Shi DQ / Qi L / Xu C / Sun R / Li XW / Lau PM ...Liu YT / Tao CL / Zhang X / Xia W / Shi DQ / Qi L / Xu C / Sun R / Li XW / Lau PM / Zhou ZH / Bi GQ
Funding support China, United States, 3 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31630030 China
National Science Foundation (NSF, United States)GM071940 United States
Ministry of Science and Technology (MoST, China)2017YFA0505300 China
CitationJournal: Nat Neurosci / Year: 2020
Title: Mesophasic organization of GABA receptors in hippocampal inhibitory synapses.
Authors: Yun-Tao Liu / Chang-Lu Tao / Xiaokang Zhang / Wenjun Xia / Dong-Qing Shi / Lei Qi / Cheng Xu / Rong Sun / Xiao-Wei Li / Pak-Ming Lau / Z Hong Zhou / Guo-Qiang Bi /
Abstract: Information processing in the brain depends on specialized organization of neurotransmitter receptors and scaffolding proteins within the postsynaptic density. However, how these molecules are ...Information processing in the brain depends on specialized organization of neurotransmitter receptors and scaffolding proteins within the postsynaptic density. However, how these molecules are organized in situ remains largely unknown. In this study, template-free classification of oversampled sub-tomograms was used to analyze cryo-electron tomograms of hippocampal synapses. We identified type-A GABA receptors (GABARs) in inhibitory synapses and determined their in situ structure at 19-Å resolution. These receptors are organized hierarchically: from GABAR super-complexes with a preferred inter-receptor distance of 11 nm but variable relative angles, through semi-ordered, two-dimensional receptor networks with reduced Voronoi entropy, to mesophasic assembly with a sharp phase boundary. These assemblies likely form via interactions among postsynaptic scaffolding proteins and receptors and align with putative presynaptic vesicle release sites. Such mesophasic self-organization might allow synapses to achieve a 'Goldilocks' state, striking a balance between stability and flexibility and enabling plasticity in information processing.
History
DepositionJul 28, 2020-
Header (metadata) releaseNov 11, 2020-
Map releaseNov 11, 2020-
UpdateDec 9, 2020-
Current statusDec 9, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0001
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.0001
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22366.png

Downloads & links

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Map

FileDownload / File: emd_22366.map.gz / Format: CCP4 / Size: 1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationreconstruction GABA receptor pair
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
8.7 Å/pix.
x 64 pix.
= 556.8 Å		8.7 Å/pix.
x 64 pix.
= 556.8 Å		8.7 Å/pix.
x 64 pix.
= 556.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 8.7 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0001 / Movie #1: 0.0001
Minimum - Maximum-0.0033990422 - 0.0031378688
Average (Standard dev.)0.00003004293 (±0.0007371973)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions646464
Spacing646464
CellA=B=C: 556.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z8.78.78.7
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z556.800556.800556.800
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ240240240
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS646464
D min/max/mean-0.0030.0030.000

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Supplemental data

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Sample components

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Entire : gamma-aminobutyric acid receptor

EntireName: gamma-aminobutyric acid receptor
Components
  • Complex: gamma-aminobutyric acid receptor

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Supramolecule #1: gamma-aminobutyric acid receptor

SupramoleculeName: gamma-aminobutyric acid receptor / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Rattus norvegicus (Norway rat) / Organ: brain / Tissue: hippocampus / Location in cell: synaptic membrane
Molecular weightTheoretical: 250 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.3
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
DetailsThis sample is cultured hippocampal neurons.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 2.5 e/Å2
Details: Tilt series were collected first from 0 to -60 and then from +2 to +60 at 2 intervals using FEI Xplore 3D software, with the defocus value set at -12 to -18 um, and the total electron dosage ...Details: Tilt series were collected first from 0 to -60 and then from +2 to +60 at 2 intervals using FEI Xplore 3D software, with the defocus value set at -12 to -18 um, and the total electron dosage of about 100 e-/A2. The final pixel size was 0.755 nm. For the analysis of GABAARs, cryoET data were collected using a Titan Krios (Thermo Fisher) equipped with a Volta phase plate (VPP), a post-column energy filter (Gatan image filter), and a K2 Summit direct electron detector (Gatan). The energy filter slit was set at 20 eV. The Titan Krios was operated at an acceleration voltage of 300 KV. When VPP was used, the defocus value was maintained at -1 um; otherwise, it was maintained at -4 um. The VPP was conditioned by pre-irradiation for 60 s to achieve an initial phase shift of about 0.3pi. Images were collected by the K2 camera in counting mode or super-resolution mode. When counting mode was used, the pixel size was 0.435 nm. For super-resolution mode image, the final pixel size was 0.265 nm. Tilt series were acquired using SerialEM49 with two tilt schemes: from +48 to -60 and from +50 to +66 at an interval of 2; from +48 to -60 and from +51 to +66 at an interval of 3. The total accumulated dose is ~150 e-/A2. For sub-tomogram analysis, 6 grids were used for data collection. Totally, 32 and 40 inhibitory synapses were imaged with and without VPP, respectively.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution method: OTHER / Software - Name: RELION
Details: Directly reconstructed using relion_reconstruct with calculated orientations based on the geometry.
Number subtomograms used: 10032
ExtractionNumber tomograms: 70 / Number images used: 307091 / Software - Name: IMOD
CTF correctionSoftware - Name: NOVACTF
Final angle assignmentType: OTHER / Software - Name: RELION
Details: Direct reconstruction using relion_reconstruct with predetermined angles

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